Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. Microtubules execute their diverse cellular roles by forming suprastructures with highly distinctive geometries: the radial cytoplasmic array, the short, highly parallel axonemal array, the spindle array or the tiled long axonal array. The microtubule cytoskeleton is a complex function of many unit operations, the individual actions of cytoskeletal regulators: nucleation, growth and shrinkage, severing and motor movement. Moreover, the microtubule itself is more than just a naive roadway for cellular components to transit along. Alpha and beta tubulins have multiple isoforms and are subject to highly diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules. Given the central role microtubules play in basic cellular processes, it is not surprising that microtubule regulators have been implicated in many human diseases, including cancers, cardiovascular disease, fungal, bacterial and viral infections, as well as neurodegenerative disorders such as Parkinson's, Alzheimer's and Amyotrophic lateral sclerosis. Our efforts concentrate on two families of microtubule regulators: microtubule severing enzymes and enzymes that post-translationally modify tubulin. Our research plan is highly interdisciplinary, integrating techniques and concepts from biophysics, structural, molecular and cell biology to answer two closely interdigitated questions: how is the structure of the microtubule locally perturbed when it is engaged by these regulators and how do these regulators affect microtubule architecture and dynamics at the cellular level? Perturbation of microtubule dynamics has emerged as a common theme in a variety of neurodegenerative diseases and our work has implications for the etiologies of all these disorders. In the last year we initiated several studies aimed at understanding the mechanistic underpinnings of the functions of microtubule post-translational modifications as well as continued our work on the mechanism of microtubule severing by spastin. We are actively working on purifying to homogeneity and in biophysical quantities several tubulin modification enzymes to investigate their mechanism of action. In addition, we have made significant progress on deciphering the biophysical mechanism of action of tubulin tyrosine ligase. Tubulin tyrosine ligase adds a C-terminal Tyr to alpha&#8722;tubulin as part of a tyrosination/detyrosination cycle present in most eukaryotic cells. This C-terminal tyrosine acts as an ON/OFF switch for the recruitment of microtubule interacting proteins. Tubulin tyrosine ligase is essential for development and cellular function and its suppression leads to formation of tubulin rich tentacles that penetrate endothelial layers to facilitate the reattachment of circulating tumor cells during metastasis, thus correlating with poor prognosis in neuroblastoma, breast and prostate cancer patients. We are now using this enzyme to modify microtubules in vitro in order to investigate the effects of the introduced tubulin modification on microtubule dynamics and the recruitment of motors and microtubule association proteins.